In bacterial and animal cells, abnormal and certain normal regulatory proteins are degraded very rapidly. This degradative process reduces the accumulation of abnormal proteins in genetic diseases (e.g. hemoglobinopathies, CF) and in aging, and is critical in the regulation of cell growth. It is now clear that molecular chaperones, in addition to catalyzing protein folding and translocation, play an important role in the selective degradation of abnormal proteins. The primary goal of these studies is to elucidate this new role of the chaperones. In E.coli, the heat-shock proteins (Hsps), dnaK (Hsp7O), DnaJ and GrpE, are necessary for the rapid degradation of certain mutant polypeptides (e.g. PhoA61), while the breakdown of the fusion protein, CRAG, requires other chaperones, GroEL, GroES, and the putative chaperone, Trigger Factor. Our hypothesis is that if chaperones fail to fold an abnormal protein, they maintain them in a conformation that facilitates digestion by the ATP- dependent proteases. Enzymological studies will attempt to reconstitute in vitro these proteolytic processes in order to clarify how the chaperones enhance the degradation of these proteins. In vivo, these short-lived proteins are found in large "degradative complexes" containing multiple chaperones. We shall investigate the composition and functional significance of these complexes in proteolysis. In eukaryotic cells, molecular chaperones are also required for degradation of certain proteins by the ubiquitin-proteasome pathway. In yeast, mutations in the DnaJ homologs, Ydj and Sis, and in Hsp7Os of the Ssa and Ssb families reduce the rapid degradation of certain abnormal proteins and short-lived normal proteins. By using chaperone-deficient mutants, we shall test if these hsps are required for the breakdown of important short-lived regulatory proteins (e.g. cyclins and transcription factors). Our current model is that chaperones bind to substrates and present them to the ubiquitinating enzymes or to the 265 proteasome. Biochemical approaches will be used to test this hypothesis, i.e. to clarify how Ydj and Ssa promote ubiquitination to certain substrates, and how Sis and Ssb facilitate degradation of ubiquitinated proteins by the 265 proteasome. These studies will be performed using pure components and extracts of chaperone-deficient mutants and also of reticulocytes to see if the chaperones play similar roles in proteolysis in mammalian cells.